Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunctions in inflammation, sepsis, and ischemic reperfusion injury. The abnormality is largely attributable to neutrophil (PMN) activation and releasing edemagenic agents. The overall goal of this research project is to define the precise molecular mechanisms of PMN adhesion-dependent and adhesion-independent microvascular barrier injury. Our studies during the prior funding cycle have revealed a series of coordinated signaling and structural reactions at the microvascular endothelium during PMN stimulation, characterized by focal adhesion-supported cytoskeleton contraction and junction disorganization. Comparative analysis of the complex pathways points to a predominant role for myosin light chain (MLC)-mediated contractile force development as a triggering event to open the paracellular pathway for protein leakage across the endothelium. As a continuing effort, this study is designed to further characterize the endothelial contractile mechanism by testing the hypothesis that PMNs induce actin polymerization and MLC phosphorylation via p60Src-activation of endothelial-specific myosin light chain kinase (eMLCK) and RhoA-inhibition of the myosin targeting subunit of myosin light chain phosphatase (MLCP). Three specific aims are proposed: 1) to study the eMLCK mechanism of PMN-elicited microvascular leakage, 2) to study the MLCP regulation of microvascular barrier function during PMN activation, and 3) to characterize the mechanical and molecular basis of endothelial contractile response in PMN-induced hyperpermeability. The research plan incorporates genetic, molecular, and pharmacological approaches into an integrative analysis of molecular reactions and barrier function at both the microvascular and subendothelial levels. Intravital microscopic studies will be complemented by in situ and in vitro experiments for a comprehensive evaluation of hydraulic conductivity and solute permeability. The study will provide novel mechanistic insights into the regulation of microvascular endothelial barrier function by PMNs, with broad implications in various types of vascular diseases or injuries.